Novozymes Lipase Products. Strem Chemicals, Inc. Storage

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1 Strem Chemicals, Inc. Catalog # Novozym Note: Sold in collaboration with Novozymes A/S for research purposes only. Novozym is a 1,3 specific lipase originating from Rhizmucor miehei which is immobilized on a resin carrier. The enzyme is a highly effective catalyst for stereoselective transesterification and ester hydrolysis. Declared activity 275 IUN/g. Lipase that hydrolyzes ester bonds in glycerides. Color can vary from batch to batch. Color intensity is not an indication of enzyme activity. Packaging must be kept intact, dry and away from sunlight. Please follow the recommendations and use the product before the best before date to avoid the need for a higher dosage. Other Lipase products offered by Strem: Novozym Lipozyme TL IM Novocor AD L Novozym Lipozyme CALB L Lipozyme TL 100 L Resinase HT Novozym Novozymes Lipase Screening Kit (contains 9 lipase enzymes) Endoprotease products offered by Strem: Alcalase 2.4 L FG Alcalase 2.5 L Savinase 12 T Savinase 16 L Esperase 8.0 L Neutrase 0.8 L Novozymes Endoprotease Screening Kit (contains 6 endoprotease enzymes) Novozymes Lipase Products Storage Kits should be optimally stored at 0-10C/32-50F. If stored above 25C/77F the samples should be used within 3 months. Introduction Lipases (EC Number ) are one of the most commonly used classes of enzymes in biocatalysis. They have been used on a variety of substrates and show very broad substrate specificity. Lipases catalyze the hydrolysis of triacylglycerols to diacylglycerol, monoacylglycerol, glycerol and free fatty acids. The reaction reverses under anhydrous conditions and the enzyme

2 is able to synthesize new molecules by esterification, alcoholysis and transesterification. All reactions can be performed with high regio- and enantioselectivity under mild reaction conditions. Figure 1: Regioselective hydrolysis of a triacylglycerol. Description and optimum usage conditions Strem Enzyme ph Temp Substrate Catalog Product Name Activity* Formulation No. optimum optimum specificity Number Novozym PLU/g Immobilized ph C Esters and alcohols Lipozyme TL IM 250 IUN/g Immobilized ph C Esters Novozym IUN/g Immobilized ph C Esters Lipozyme CALB L 5000 LU/g Liquid ph C Esters and alcohols Palatase L LU/g Liquid ph C Esters Lipozyme TL 100 L 100 KLU/g Liquid ph C Esters and diesters NovoCor AD L 6000 LU/g Liquid ph C Sterically hindered esters Resinase HT 50 KLU/g Liquid ph 5-8 up to 90C Esters Novozym KLU/g Liquid ph C Esters * K = Kilo, LU = Lipase unit, PLU = Propyl Laurate Unit, IUN = Interesterification Unit. 1LU is the amount of enzyme activity which liberates 1 µmol of tritratable butyric acid from the substrate glycerol tributyrate per minute under defined standard conditions. 1LU is equal to 1IUN. 1 PLU is the amount of enzyme activity which generates 1 µmol of propyl laurate per minute under defined standard conditions.

3 Kinetic Resolution Example 1. Kinetic resolution by transesterification of racemic alcohol 1 Racemic alcohol (1-2 mmol) is solubilized in organic solvent (10 ml Toluene (dry) or other solvent)* Acyl donor vinyl acetate (1:3 or 1:5 molar ratio compared to racemic alcohol) is added. Immobilized Lipase Enzyme1-3 (50% wt/wt with regards to substrate) is added and the reaction is conducted under stirring. Typical reaction temperature is o C and typical reaction time is hours, The reaction product is recovered by removing the immobilized enzyme by filtration. * Alternative solvents: methyl tert butyl ether (MTBE), n-hexane, iso-octane. Example 2. Kinetic resolution of racemic alcohol by hydrolysis of acetoxy derivative Racemic acetoxy ester (1-2 mmol) is solubilized/dispersed in potassium phosphate buffer (0.1 M, ph 7.5, 10 ml). For liquid substrates, emulsion or suspension will be formed. For solid substrates, solution is prepared by adding 10% v/v of organic solvent* Lipase Enzyme 1-9 is added to the substrate solution (50% wt/wt for solid enzyme or 10-20% v/v with regards to buffer for liquid enzyme) Reaction mixture ph is maintained at 7.5 by adjusting with 1N NaOH. Typical reaction temperature is C and typical reaction time is hours. The reaction product is recovered by extraction or filtration. *Solvent examples: IPA, acetone, tert-butanol, THF or acetonitrile

4 2, 3, 4 Example 3. Dynamic kinetic resolution of racemic alcohols Ruthenium catalyst* (0.05 eq with regards to substrate), Immobilized lipase 1-3 (50% w/w with regards to substrate) and Na 2 CO 3 (1.0 eq. with regards to substrate) are dissolved in dry organic solvent (5 ml Toluene**) under inert atmosphere (N 2 ) in a closed vessel. Dry toluene is added and resulting mixture is stirred. A THF solution of t-buok (0.05 eq with regards to substrate) is added to reaction mixture. After 30 min. of stirring, racemic alcohol (1-2 mmol) dissolved in toluene (5 ml) is added at C and stirring continued in 10 min. Vinyl acetate (3.0 eq with regards to substrate) is charged to reaction mixture at C. Reaction temperature is C and typical reaction time is 36 hours. Reaction product is recovered by filtration through filter aid and the filtrate is concentrated to obtain crude product. * Ruthenium catalyst options: Chlorodicarbonyl (1,2,3,4,5-pentaphenylcyclopentadienyl) ruthenium (η 5 C 5 Ph 5 )Ru(CO) 2 Cl or Shvo catalyst - 1-Hydroxytetraphenylcyclopentadienyl-(tetraphenyl-2,4-cyclopentadien-1- one)-μ-hydrotetracarbonyldiruthenium(ii) ** Solvents have to be dried before using (moisture content should be < 0.01%) Example 4. Kinetic Resolution by transesterification of Racemic Amine 5, 6 Acyl donor* ethyl acetate (5 ml) is charged to vessel under inert atmosphere (N 2 ) Racemic amine (1-2 mmol) and Immobilized lipase 1-3 (50% wt/wt with regards to substrate) is added to ethyl acetate at o C under moderate stirring and inert atmosphere. Typical reaction temperature is C and typical reaction time is hours, Reaction product is recovered by filtration, whereby immobilized enzyme is removed. *Acyl donor solvent: α-methylbenzyl acetate, methylmethoxy acetate, ethyl acetate and methyl tert butyl ether.

5 Example 5. Kinetic resolution by hydrolysis of racemic carboxylic ester Racemic ester (1-2 mmol), organic solvent (5 ml MTBE or Toluene) and potassium phosphate buffer (0.1 M, ph 7.0, 5 ml) is homogenized by stirring. [Two layers will form once stirring is stopped; stir until substrate is soluble in organic phase. In case of immobilized enzymes, solid suspension is observed.] Lipase Enzyme (50% wt/wt with regards to substrate for solid enzyme 1-3 or 10-20% v/v with regards to solvent mixture for liquid enzyme 4-9) is added under stirring. Reaction mixture is maintained at ph 7.0 by adjusting with 1N NaOH. Typical reaction temperature is o C and typical reaction time is hours, Reaction product is recovered by extraction or filtration. Example 6. Desymmetrisation of diesters 7, 8 Racemic diester (1-2 mmol) and potassium phosphate buffer (0.1 M, ph 7.0, 10 ml) is homogenized by stirring. o For liquid substrates emulsion or suspension will be formed. o For solid substrates a solution is prepared by adding additional solvents such. o Biphasic reactions can be carried out by making solution in MTBE or toluene. o Solvent free reactions can be carried out in a solid suspension. Lipase Enzyme (50% wt/wt for solid enzyme 1-3 or 10-20% v/v with regards to buffer for liquid enzyme 4-9) is added and stirring continued. Reaction mixture is maintained at ph 7.5 by adjusting with 1N NaOH. Typical reaction temperature is o C and typical reaction time is hours, Reaction product is recovered by extraction or filtration. *Solvent: acetone, tetrahydrofuran (THF) or acetonitrile.

6 Analytical Method Principles In-process reaction monitoring: Depending on substrate and product, different methods can be used for in process reaction monitoring. Thin Layer Chromatography (TLC) is a simple method for monitoring reaction progress and completion. To quantitatively estimate product formation and consumption of reactant, HPLC or GC can be used for monitoring. Chiral HPLC is recommended to estimate chiral purity or consumption of isomers of racemic mixture. Final chiral purity can be obtained by analyzing product isolated by using an appropriate chiral column. Key parameters for Enantiomeric excess (ee) and Enantioselectivity (E) can be calculated from the areas in chiral HPLC: % ee = ((R-S)/(R+S)) 100 where R and S stand for the individual optical isomer in the mixture (and R +S = 1) Where R = area for R isomer and S = area for S isomer Screening Procedure Listed below is recommended equipment for conducting the screens, however, ph-stat system gives more consistent results. Simple equipment Reaction vessel (25 ml round bottom or Erlenmeyer flask or test tubes) ph-meter or ph-paper (range 5-9) Burette or calibrated addition funnel Propeller mixer or magnetic needle Advanced equipment Thermostated reaction vessel (25 ml) Autotitrator/pH-stat system (ph-meter, automatic burette/addition funnel) Recording device (e.g., x/y-plotter) Propeller mixer

7 Buffer Preparation 0.1M Potassium Phosphate Buffer at 25 o C 0.1M Sodium Phosphate Buffer at 25 o C ph Volume of 1M K 2 HPO 4 (ml) Volume of 1M KH 2 PO 4 (ml) ph Volume of 1M Na 2 HPO 4 (ml) Volume of 1M NaH 2 PO 4 (ml) Dilute combined 1M stock solutions to 1 L with distilled H 2 O. References Dilute combined 1M stock solutions to 1 L with distilled H2O. 1. H. V. Ferreira, L. C. Rocha, R.P. Severino and André L. M. Porto Molecules 2012, 17, A.Traff, R. Lihammar, and J.E. Backvall J. Org. Chem. 2011, 76, Mahn-Joo Kim, Yangsoo Ahn and Jaiwook Park Current Opinion in Biotechnology 2002, 13: Kiwon Han, Cheolwoo Kim, Jaiwook Park and Mahn-Joo Kim J. Org. Chem. 2010, 75, Javier Gonzalez-Sabın, Vicente Gotor and Francisca Rebolledo Tetrahedron: Asymmetry 16 (2005) Mahn-Joo Kim, Won-Hee Kim, Kiwon Han, Yoon Kyung Choi, and Jaiwook Park Org. Lett., 9, No. 6, M. J. Homann, R. Vail, B. Morgan, V. Sabesan, C. Levy, D. R. Dodds, A. Zaks, Adv. Synth. Catal. 2001, 343, A. Goswami & T.P.Kissick Org. Proc. Res. Dev. 2009, 13, 483 The products and services described in this document are the responsibility of Novozymes Biopharma DK A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark (company registration no ) - a wholly owned subsidiary of Novozymes A/S. The information in this document is based on data we believe to be reliable. They are offered in good faith, but without warranty, as conditions and methods of use of the products are beyond our control. Furthermore, laws, regulations, and/or third-party rights may prevent the recipient from using the information herein in a given manner. Thus, the information contained herein is provided AS IS and Novozymes makes no representation or warranty whatsoever with regard to said information, hereunder the accuracy, fitness for a particular purpose, noninfringement of intellectual property rights, or regulatory/legal compliance, unless otherwise agreed in writing.